Acute respiratory distress syndrome (ARDS) can occur in patients from multiple etiologies. A hallmark of this syndrome is lung injury and edema which results in impaired oxygenation. Alveolar edema clearance needs to occur for the patients to improve. Recently, it has been recognized that the mechanisms responsible for alveolar fluid clearance in patients with ARDS are impaired, which is associated with increased mortality. Alveolar fluid clearance is affected mostly by vectorial Na+ transport, via apical Na+ channels and basolateral Na,K-ATPase in the alveolar epithelium. Thus, a better understanding of the regulation of active Na+ transport may lead to new therapeutic strategies to improve edema clearance and reduce mortality of patients with ARDS. In previous cycles of this grant, we have determined that upregulation of the (-2 and (-1 subunits of the Na+ pump increased Na,K-ATPase function in alveolar epithelial cells (AEC) and increased lung edema clearance. We have also determined that G-protein coupled receptors (GPCR) agonists increased Na,K-ATPase function which lead to increased alveolar fluid clearance. However, the mechanisms regulating GPCR-mediated upregulation of the Na,K-ATPase have not been fully elucidated. In this current proposal we will focus on the mechanisms regulating the GPCR mediated increase in Na,K-ATPase function and alveolar fluid reabsorption via three interrelated specific aims: in studies proposed for Specific Aim 1 we will determine whether the salt inducible kinase (SIK) participates in the GPCR mediated Na,K-ATPase upregulation by facilitating the traffic of Na+ pump molecules from intracellular pools into the AEC plasma membrane; in studies pertaining to Specific Aim 2, we will determine the role of actin-based molecular motors and specifically the role of the myosin V family in the traffic of Na,K-ATPase vesicles and thus Na,K-ATPase upregulation via its recruitment to the plasma membrane of alveolar epithelial cells; and in experiments pertaining to Specific Aim 3, we will determine whether the intracellular trafficking of Na,K-ATPase during recruitment to the plasma membrane is regulated by the cytoskeleton-microtubule molecular motors, kinesin and dynein. Experiments have been conducted for each of the specific aims and the preliminary data support the feasibility of the proposed experiments. The data generated from the proposed studies will provide important new information on the role and regulation of Na,K-ATPase and active Na+ transport in rodent lungs which will help with the design of new strategies to increase lung edema clearance in patients with the acute respiratory distress syndrome. Relevance to Public Health: Patients with acute respiratory distress syndrome (ARDS) have lung injury and fluid (edema) accumulation in their airspaces. This fluid in their lungs precludes them from breathing normally as it impairs the transfer of oxygen from the room through the lungs and into all the organs of the human body. For the patient with ARDS to survive the fluid from the lungs needs to be cleared. The protein sodium/potasium adenyl triphosphatase (Na,K-ATPase) has an important role in fluid clearance from the lungs such that when more Na,K-ATPase molecules are present at the lung epithelial cell membrane it increases fluid clearance from normal and injured lungs. In this grant application we propose to study mechanisms by which Na,K-ATPase can be increased in the lung epithelial cell membranes leading to increased fluid clearance from the lungs. The results from the proposal should be of importance to design novel strategies to treat patients with ARDS such as the ones conducted by the ARDSnet.